Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23697
標題: 以Bacillus-Yeast共培養策略建構纖維素乙醇生產系統
Construction of cellulosic ethanol production process via Bacillus-yeast co-culturing strategy
作者: 何政育
Ho, Cheng-Yu
關鍵字: artificial cellulosome;人工纖維素分解酵素複合體;co-culture;cellulosic ethanol;共培養;纖維乙醇
出版社: 生命科學系所
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摘要: 
Kefir酵母菌Kluyveromyces marxianus KY3具有很廣泛的基質利用性、很好的高溫發酵潛力及優異的異源基因表現能力。我們建構了一個以酵母菌為宿主的合成生物學技術,可以同時轉入多個基因於宿主染色體中,我們稱它為Promoter-based Gene Assembly and Simultaneous Overexpression〔PGASO〕,此方法利用特定的推動子重疊區域作為多基因同源重組的依據,PGASO已成功應用於酵母菌K. marxianus KY3,當我們同時轉入五個基因片段時,呈現相當高的轉形效率約63%。另外,K. marxianus KY3-NpaBGS帶有牛胃真菌β-glucosidase (NpaBGS)基因,這兩個基因轉殖菌種將被用在後續乙醇生產系統中。
『可設計的纖維素分解酵素複合體』是一個製作人工纖維素分解酵素複合體的重要議題,可以不同的酵素比例來面對不同的木質纖維素降解情況,參考前人針對Clostridium thermocellum ATCC27405的蛋白質體學之研究,設計了面對結晶狀纖維素(Avicel)與纖維雙糖(cellobiose)的兩種操作組以仿生式模擬C. thermocellum分解纖維質時的原始表現狀態於枯草桿菌168中表現,八個C. thermocellum的纖維素分解酵素複合體相關基因:一個支架蛋白(cipA)、一個細胞膜上連接蛋白(sdbA)、兩個纖維外切酵素(celK and celS)、兩個內切酵素(celA and cel R)、兩個聚木糖分解酵素(xynC and xynZ),利用方法Ordered Gene Assembly in B. subtilis method (OGAB)共同表現於枯草菌中。
為達成高效纖維乙醇生產製程,本研究建構了一個新的Bacillus /yeast共培養系統,兩個基轉酵母菌(KY3-NpaBGS and KR5)可以分泌纖維素分解酵素,用來與B. subtilis type I共培養,以提高纖維素糖化及發酵效率。所有Bacillus /yeast共培養系統皆可表現優於單獨CBP菌種KR5的同步纖維素糖化及乙醇轉化效率。由結果顯示,以β-glucan為碳源時,共培養組合KY3-NpaBGS+Bacillus Type I可以分別呈現6倍、34%、2.5倍的生質乙醇產量之增加。由我們的結果顯示參與菌種之特性賦予Bacillus /yeast共培養系統之纖維乙醇生產能力,且具有很好的潛力應用於同步糖化發酵的製程。

The kefir yeasts that produce ethanol and higher aromatic alcohols not only create kefir’s distinctive characteristics but could also serve as a good cell-based bioethanol production and biorefinery platform. This Kluyveromyces marxianus KY3 strain possesses a broad spectrum of substrate utilization, has a high potential for bioethanol production at elevated temperatures and suitable for expressing heterologous genes. We developed one synthetic biology techniques to transform genes into the host genome, named Promoter-based Gene Assembly and Simultaneous Overexpression 〔PGASO〕, which employed overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual unique promoters. As an example of application, PGASO was used to engineer yeast K. marxianus KY3. Our data showed high transformation efficiency and accuracy because ~63% of the transformants were found to carry the correct five-gene cassette assembly. And K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus. The two transgenic strains were employed to process ethanol production.
“Designer cellulosomes” is a concept for making an artificial cellulosome that can be proposed as a tool for regulating the cellulosomal enzyme ratio to apply on different lignocellulose degradation. Two types of “designer operon” were expressed in Bacillus subtilis 168 via biomimetic approach according to a proteome-wide analysis of Clostridium thermocellum ATCC27405, which induced by avicel and cellobiose respectively. Eight celulosomal genes including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exoglucosidase genes (celK and celS), two endoglucanase genes (celA and cel R), and two xylanase genes (xynC and xynZ) of C. thermocellum were cloned and co-expressed on the polycistronic operons in desire order via an Ordered Gene Assembly in B. subtilis method (OGAB).
A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. Two engineered yeasts, KY3-NpaBGS and KR5, which possess secretive cellulolytic enzymes, were used to co-cultivate with the B. subtilis type I strain for improving the cellulose digestion and fermentation efficiency. All Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. In our result, the co-culturing of KY3-NpaBGS with Bacillus Type I appeared 6 times, 34% and 2.5 times ethanol production increasing than KY3+TypeI, KR5+ Type I and KY3-NpaBGS+host when utilized β-glucan as carbon source. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.
URI: http://hdl.handle.net/11455/23697
其他識別: U0005-1506201209502600
Appears in Collections:生命科學系所

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